Griffin:Antigen Retrieval Technique

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Antigen Retrieval is a proven and effective method for optimizing tissue antigen detection of formalin-fixed, paraffin-embedded tissue sections. Pretreatment with an antigen retrieval reagent (Antigen unmasking) that reverses protein cross-linkage formed by formalin fixation allows greater exposure for the antibody to associate with antigenic sites. Heating methods (Microwave Oven, Pressure Cooker or Steamer/Rice Cooker) are common and effective. Below are a few effective methods to consider.

Heat Treatment

A standard Rice cooker is an effective heat/steam source for antigen unmasking

10-20 minute heat treatment is a common range that is widely effective. The cooling process taking ~20 minutes. The main mechanism of heat-induced antigen retrieval is disruption of the cross-links and pH is also an essential factor for proper refolding of epitopes. For this reason a low pH and high pH buffer is a good contrast in optimizing the staining protocol.

1) Temperature of retrieval solution should be around 95 °C.

2) Incubation time should be at least 10 minutes and it is usually around 20 minutes.

3) pH value of retrieval solution is depending on which solution you are using.

Below are 3 options to consider;

10 mM sodium citrate buffer, pH 6.0

Summary: Heat at 95º C for 5 minutes. Top off with fresh buffer and heat at 95º C for 5 minutes (optimal incubation time may vary for each tissue type). Allow slides to cool in the buffer for approximately 20 minutes. Wash in deionized H2O three times for 2 minutes each. Aspirate excess liquid from slides.

Procedure:

Materials: Citrate or Glycine-EDTA buffer, a microwaveable plastic container with slide holder and a microwave with a rotating dish.

I) For consistency, fill the slide holder with slides even if only a couple of slides with tissues are being stained. Use clean slides with no tissue
for that.

II) Fill the container with the citrate buffer, which is kept at ambient temperature.

III) Place the container with the slides and the buffer in the microwave and bring the buffer to a boil with the microwave at full power. This may take
around 2 minutes depending on the microwave used. As soon as the buffer begins to boil the power is reduced to about 30% to allow intermitent boiling of the buffer. Keep the slides for 7 minutes under these conditions. This is done so the tissues don't come off the slides due to continuous boiling. Note: the amount of power used for this step will need to be determined for each microwave.

IV) The container is left at ambient temperature to cool down.

V) Once the buffer is near ambient temperature the slides with tissues are removed, washed briefly with 1xPBS and we proceed with the peroxidase
blocking and antibody staining.

50 mM glycine-HCl buffer, pH 3.5, with 0.01% (w/v) EDTA

Summary: Heat at 95º C for 5 minutes. Top off with fresh buffer and heat at 95º C for 5 minutes (optimal incubation time may vary for each tissue type). Allow slides to cool in the buffer for approximately 20 minutes. Wash in deionized H2O three times for 2 minutes each. Aspirate excess liquid from slides.

Procedure:

Materials: Citrate or Glycine-EDTA buffer, a microwaveable plastic container with slide holder and a microwave with a rotating dish.

I) For consistency, fill the slide holder with slides even if only a couple of slides with tissues are being stained. Use clean slides with no tissue
for that.

II) Fill the container with the citrate buffer, which is kept at ambient temperature.

III) Place the container with the slides and the buffer in the microwave and bring the buffer to a boil with the microwave at full power. This may take
around 2 minutes depending on the microwave used. As soon as the buffer begins to boil the power is reduced to about 30% to allow intermitent boiling of the buffer. Keep the slides for 7 minutes under these conditions. This is done so the tissues don't come off the slides due to continuous boiling. Note: the amount of power used for this step will need to be determined for each microwave.

IV) The container is left at ambient temperature to cool down.

V) Once the buffer is near ambient temperature the slides with tissues are removed, washed briefly with 1xPBS and we proceed with the peroxidase
blocking and antibody staining.